Production of Stilbenoids

ABSTRACT

A method for the production of a stilbenoid, such as resveratrol or pinosylvin, by fermenting plant material such a grape must using a yeast having a metabolic pathway producing said stilbenoid, separating a solids waste material from said fermentation and extracting said stilbenoid.

The present invention relates to the production of stilbenoids byextraction thereof from wine making waste.

A number of strains of yeast which have been genetically engineered toproduce one or more stilebenoids such as resveratrol and pinosylvin havebeen described. Thus, South African Patent 2004/8194 (University ofStellenbosch) and Becker et al disclosed a Saccharomyces cerevisiae forfermenting wine must having introduced therein a coumarate-coenzyme-Aligase encoding gene (4CL216) and a grapevine resveratrol synthase gene(vstl). WO2006/089898 disclosed a Saccharomyces cerevisiae also havingintroduced therein a gene encoding a phenylalanine or tyrosine-ammonialyase (PAL/TAL) and a gene encoding a cinnamate 4-hydroxylase (C4H).WO2006/125000 discloses oleaginous cells having resveratrol productioncapacity. WO2008/009728 discloses a pinosylvin producing Saccharomycescerevisiae having introduced therein a PAL, 4-coumarate CoA-ligase orcinnamate-CoA ligase, a pinosylvin synthase.

The extraction of resveratrol from grape seed and skin (marc) of redwine grapes has been proposed (Australian Harvest—Press release).

Beeckwilder et al disclose resveratrol production by a geneticallyengineered yeast and detection of resveratrol in the liquid culturemedium. It was stated that resveratrol accumulated in the medium ratherthan in the cells.

Pretorius disclosed the possibility of developing wine yeasts producingresveratrol but indicated that the chances of success were unknown.

Becker et al disclosed a genetically modified yeast which producedglycosylated resveratrol during fermentation of a culture mediumcontaining coumaric acid, and discussed the possibility of fermentinggrapes with such a modified yeast in the hope of producing winecontaining more than a normal level of resveratrol. Glycosylatedresveratrol production was demonstrated by extraction from yeast cellsgrown in culture medium.

Further sources of stilbenoids are desirable. There is now providedaccording to the present invention a method for the production of astilbenoid, comprising extracting said stilbenoid from a solids wastematerial separated from a fermentation of plant material conducted usinga yeast having a metabolic pathway producing said stilbenoid.

The term ‘solids waste material’ refers to a waste material containingundissolved solids, optionally with significant quantities of freeliquid such that the waste material may be flowable or not, includingpastes or slurries as well as embracing dry solids. ‘Waste material’includes any material that is not the desired end product of thefermentation (which will typically be wine or beer of some form) andincludes residual plant material mixed with yeast cells (live or dead).

The method may further comprise the preliminary steps of conducting saidfermentation of plant material using a said yeast having a metabolicpathway producing said stilbenoid and separating a solids waste materialfrom said fermentation. The solids waste material may comprise yeastsolids and plant material solids.

Preferably, the fermentation is a fermentation of fruit must togetherwith or separated from pommace or is a fermentation of pommace separatedfrom fruit must. The fruit may for instance be grape, apple or pear. Thefermentation may be a beer making fermentation, all forms of beer beingincluded, whether obtained by the use of a top fermenting yeast or abottom fermenting yeast.

Methods for extracting stilbenoids, including resveratrol andpinosylvin, are described in the above publications. Suitable solventsinclude esters such as ethyl acetate or a solvent as described in GB0714671.5, i.e. an ester which preferably is of the general formulaR⁶—COO—R⁷, and R⁶ is H or an aliphatic straight or branched chainhydrocarbon moiety of from 1-6 carbon atoms and R′ is an aliphaticstraight or branched chain hydrocarbon moiety of from 2-16 carbon atoms,or a heteroatom containing hydrocarbon moiety of from 2 to 16 carbonatoms or an aromatic or heteroaromatic moiety of from 5 to 16 carbonatoms. R⁷ may have from 3 to 9 carbon atoms. R⁶ may have from 1 to 4carbon atoms.

Preferably, said ester is an octyl acetate, especially n-octyl acetate.

Optionally, said liquid comprises or further comprises an alkane. It mayconsist of a said alkane and a said ester. Said alkane may be a C₆ toC₁₆ straight or branched chain alkane, e.g. a C₉₋₁₄ alkane, e.g. a C 2alkane. Preferably, said alkane is n-dodecane.

The stilbenoid producing yeast may be as described in any of the abovepublications or genetically engineered according to the principles orpractice there described. In particular, it may be a resveratrolproducing yeast as described generally or by way of example inWO2006/089898 or a pinosylvin producing yeast as described generally orby way of example in WO2008/009728.

Preferably therefore, the yeast may be one having an operative metabolicpathway comprising at least one enzyme activity, said pathway producing4-coumaric acid and producing resveratrol therefrom, or an oligomeric orglycosidically-bound derivative thereof preferably by a reactioncatalysed by an enzyme in which endogenous malonyl-CoA is a substrate.Preferably the resveratrol is produced from 4-coumaroyl-CoA by aresveratrol synthase expressed in said micro-organism from nucleic acidcoding for said enzyme which is not native to the yeast.

The 4-coumaric acid may be produced from trans-cinnamic acid by acinnamate 4-hydroxylase not native to the yeast.

4-coumaric acid may be produced from tyrosine in a reaction catalysed bya L-phenylalanine ammonia lyase or a tyrosine ammonia lyase not nativeto the yeast. Trans-cinnamic acid may be produced from L-phenylalaninein a reaction catalysed by a L-phenylalanine ammonia lyase not native tothe yeast. 4-coumaroyl-CoA may be formed in a reaction catalysed by a4-coumarate-CoA ligase introduced into the yeast.

A native NADPH:cytochrome P450 reductase (CPR) may be expressed in theyeast or may recombinantly introduced.

Thus, the yeast may be one containing one or more copies of anheterologous DNA sequence encoding phenylalanine ammonia lyaseoperatively associated with an expression signal, and containing one ormore copies of an heterologous DNA sequence encodingcinnamate-4-hydroxylase operatively associated with an expressionsignal, and containing one or more copies of an heterologous DNAsequence encoding 4-coumarate CoA-ligase operatively associated with anexpression signal, and containing one or more copies of an heterologousDNA sequence encoding resveratrol synthase operatively associated withan expression signal, or may be one lacking cinnamate-4-hydroxylaseactivity, and containing one or more copies of a heterologous DNAsequence encoding tyrosine ammonia lyase operatively associated with anexpression signal, and containing one or more copies of an heterologousDNA sequence encoding 4-coumarate CoA-ligase operatively associated withan expression signal, and containing one or more copies of anheterologous DNA sequence encoding resveratrol synthase operativelyassociated with an expression signal.

For the production of pinosylvin, the yeast may have an operativemetabolic pathway comprising at least one enzyme activity, said pathwayproducing pinosylvin from cinnamic acid and preferably producingcinnamic acid and produces pinosylvin therefrom. Said pinosylvin may beproduced in a reaction catalysed by an enzyme in which endogenousmalonyl-CoA is a substrate, suitably from cinnamoyl-CoA by a stilbenesynthase, suitably expressed in the yeast from nucleic acid coding forsaid enzyme which is not native to the yeast.

Cinnamic acid is preferably produced in said pathway fromL-phenylalanine in a reaction catalysed by a L-phenylalanine ammonialyase (PAL) which may be not native to the yeast.

Said PAL is preferably one accepting phenylalanine as a substrate andproducing cinnamic acid therefrom, such that if the PAL also acceptstyrosine as a substrate and forms coumaric acid therefrom, the ratio

Km(phenylalanine)/Km(tyrosine) for said PAL is less than 1:1 andpreferably such that the ratio K_(cat)(PAL)/K_(cat)(C4H) is at least2:1.

Cinnamoyl-CoA may be formed in a reaction catalysed by a 4-coumarate-CoAligase or a cinnamoyl-CoA ligase which may be not native to the yeast.

Any or all of at least one copy of a genetic sequence encoding aphenylalanine ammonia lyase, at least one copy of a genetic sequenceencoding a 4-coumarate-CoA ligase or cinnamate-CoA ligase, at least onecopy of a genetic sequence encoding a resveratrol synthase or apinosylvin synthase may be present operatively linked to an expressionsignal not natively associated with said genetic sequence.

Thus the yeast may be one containing one or more copies of anheterologous DNA sequence encoding phenylalanine ammonia lyaseoperatively associated with an expression signal, and containing one ormore copies of an heterologous DNA sequence encoding 4-coumarateCoA-ligase or cinnamate-CoA ligase operatively associated with anexpression signal, and containing one or more copies of an heterologousDNA sequence encoding resveratrol synthase operatively associated withan expression signal or may be one containing one or more copies of anheterologous DNA sequence encoding phenylalanine ammonia lyaseoperatively associated with an expression signal, and containing one ormore copies of an heterologous DNA sequence encoding 4-coumarateCoA-ligase or cinnamate-CoA ligase operatively associated with anexpression signal, and containing one or more copies of an heterologousDNA sequence encoding pinosylvin synthase operatively associated with anexpression signal.

In all cases, expression of the gene ACC1 may be boosted to increase thepool of malonyl-CoA available in the metabolic pathway.

The yeast may be of the genus Saccharomyces and may be of the speciesSaccharomyces cerevisiae, S. kluyveri, S. bayanus, S. exiguus, S.sevazzi, or S. uvarum or others, especially any conventionally used inbrewing or wine making.

The accompanying drawings show results obtained in the examples asfollows:

FIG. 1 shows a divergent fusion fragment formed between a TEF2 promoterand TDH3 promoter;

FIG. 2 shows HPLC chromatograms of pulp extract obtained in Example 14with Absorbance plotted against retention time;

FIG. 3 shows further chromatograms obtained in Example 14 showing UVspectra of pinosylvin peaks in waste stream extracts (pulp sample andsediment sample) and of a standard; and

FIG. 4 shows still HPLC chromatograms of sediment obtained in Example 14with Absorbance plotted against retention time.

The invention will be illustrated by the following non-limitingexamples.

EXAMPLES Example 1 Isolation of Genes Encoding PAL2, C4H, AR2, 4CL andVST1

Phenylalanine ammonia lyase (PAL2) (Cochrane et al., 2004) (SEQ ID NO1), cinnamate 4-hydroxylase (C4H) (Mizutani et al, 1997) (SEQ ID NO 2),cytochrome P450 reductase (AR2)(Mizutani and Ohta, 1998) (SEQ ID NO 3),4-coumarate:coenzymeA ligase (4CL) (Hamberger and Hahlbrock 2004;Ehlting et al., 1999) (SEQ ID NO 4) were isolated via PCR from A.thaliana cDNA (BioCat, Heidelberg, Germany) using the primers in table1.

The codon optimized VST1 gene encoding Vitis vinifera (grapevine)resveratrol synthase (Hain et al., 1993) (SEQ ID NO 5) for expression inS. cerevisiae was synthesized by GenScript Corporation (Piscataway,N.J.). The synthetic VST1 gene was delivered inserted in E. coli pUC57vector flanked by BamH1 and Xho1 restriction sites. The synthetic genewas purified from the pUC57 vector by BamH1/Xho1 restriction andpurified from agarose gel using the QiaQuick Gel Extraction Kit(Qiagen).

TABLE 1  Primer for amplification of gene(Restriction sites are underlined) Gene5-CG GAATTC CGTACG TA ATG GAT CAA ATC PAL2 GAA GCA ATG TT SEQ ID NO: 105-CG ACTAGT TTA GCA AAT CGG AAT CGG AGC PAL2 SEQ ID NO: 115-CG CTCGAG GCGGCCGC TAAAAT ATG GAC CTC C4H CTC TTG CTG GAGSEQ ID NO: 12 5-AGTAGAIGGAGTAGATGGAGTAGATGGAGTAGATGG C4HACA GTT CCT TGG TTT CAT AAC G SEQ ID NO: 135-CCATCTACTCCATCTACTCCATCTACTCCATCTACT AR2 AGG AGA TCC GGT TCT GGG ASEQ ID NO: 14 5-CG GGTACCAT TTA CCA TAC ATC TCT AAG AR2 ATA TCT TCCSEQ ID NO: 15 5′ GCGAATTCTTATGACGACACAAGATGTGATAGTCAA 4CL TGATSEQ ID NO: 16 5′ GCACTAGTATCCTAGTTCATTAATCCATTTGCTAG 4CL TCTTGCSEQ ID NO: 17

The coding sequence of tyrosine ammonia lyase (TAL) from Rhodobactercapsulatus (Kyndt et al., 2002; is codon optimized for expression in S.cerevisiae using the online service backtranslation tool atwww.entelechon.com, yielding sequence SEQ ID NO: 6)

Example 2 Construction of a Yeast Vector for Galactose InducedExpression of PAL2 and C4H:AR2 Fusion Gene

The gene encoding PAL2 was amplified from cDNA from A. thaliana astemplate using forward primer 5-CG GAATTC CGTACG TA ATG GAT CAA ATC GAAGCA ATG TT-3 SEQ ID NO 29 and reverse primer 5-CG ACTAGT TTA GCA AAT CGGAAT CGG AGC-3 SEQ ID NO 30. The amplified PAL2 PCR-product was digestedwith EcoR1/Spe1 and ligated into EcoR1/Spe1 digested pESC-URA vector(Stratagene), resulting in vector pESC-URA-PAL2. Two different clones ofpESC-URA-Pal2 were sequenced to verify the sequence of the cloned gene.

C4H was amplified using cDNA from A. thaliana as template using forwardprimer 5-CG CTCGAG GCGGCCGC TAAAAT ATG GAC CTC CTC TTG CTG GAG-3 SEQ IDNO 31 and reverse primer 5-AGTAGATGGAGTAGATGGAGTAGATGGAGTAGATGG ACA GTTCCT TGG TTT CAT AAC G-3 SEQ ID NO 32. AR2 was amplified using cDNA fromA. thaliana as template using forward primer5-CCATCTACTCCATCTACTCCATCTACTCCATCTACT AGG AGA TCC GGT TCT GGG A-3 SEQID NO 33 and reverse primer 5′-CG GGTACCAT TTA CCA TAC ATC TCT AAG ATATCT TCC-3 SEQ ID NO 34. The amplified PCR products C4H and AR2 were usedas templates for the creation of the fusion gene C4H:AR2 using theforward primer 5-CG CTCGAG GCGGCCGC TAAAAT ATG GAC CTC CTC TTG CTG GAG-3SEQ ID NO 35 and the reverse primer 5-CG GGTACC AT TTA CCA TAC ATC TCTAAG ATA TCT TCC-3 SEQ ID NO 36.

The fusion gene C4H:AR2 gene was digested with XhoI/KpnI and ligatedinto XhoI/KpnI digested pESC-URA-PAL2. The resulting plasmid,pESC-URA-PAL2-C4H:AR2, contained the genes encoding PAL2 and C4H:AR2under the control of the divergent galactose induced <=GAL1/GAL10=>promoters. The sequence of the gene encoding C4H:AR2 was verified bysequencing of two different clones of pESC-URA-PAL2-C4H:AR2.

Example 3 Construction of a Yeast Vector for Galactose InducedExpression of 4CL1 and VST1

The gene encoding 4CL was isolated as described in example 1. Theamplified 4CL PCR-product was digested with EcoR1/Spe1 and ligated intoEcoR1/Spe1 digested pESC-HIS vector (Stratagene), resulting in vectorpESC-HIS-4CL.

Two different clones of pESC-HIS-4CL were sequenced to verify thesequence of the cloned gene.

The gene encoding VST1 was isolated as described in example 1. Theamplified synthetic VST1 gene was digested with BamH1/Xho1 and ligatedinto BamH1/Xho1 digested pESC-HIS-4CL. The resulting plasmid,pESC-HIS-4CL-VST1, contained the genes encoding 4CL and VST1 under thecontrol of the divergent galactose induced <=GAL1/GAL10=> promoters. Thesequence of the gene encoding VST1 was verified by sequencing of twodifferent clones of pESC-HIS-4CL-VST1.

Example 4 Construction of Strong Constitutive Promoter Fragment TDH3

The 600 base pair TDH3 (GPD) promoter was amplified from S. cerevisiaegenomic DNA using the forward primer 5′GC GAGCTC AGT TTA TCA TTA TCA ATACTC GCC ATT TCA AAG SEQ ID NO: 18 containing a Sac1 restriction site andthe reverse primer 5′-CG TCTAGA ATC CGT CGA AAC TAA GTT CTG GTG TTT TAAAAC TAA AA SEQ ID NO: 19 containing a Xba1 restriction site. Theamplified TDH3 fragment was digested with Sac1/Xba1 and ligated intoSac1/Xba1 digested plasmid pRS416 (Sikorski and Hieter, 1989) asdescribed previously (Mumberg et al, 1995) resulting in plasmidpRS416-TDH3.

Example 5 Construction of Constitutive Strong Promoter Fragment TEF2

The 400 base pair TEF2 promoter was amplified from S. cerevisiae genomicDNA using the forward primer 5′-GC GAGCTC ATA GCT TCA AAA TGT TTC TACTCC TTT TTT ACT CTT SEQ ID NO: 20 containing a Sac1 restriction site andthe reverse primer 5′-CG TCTAGA AAA CTT AGA TTA GAT TGC TAT GCT TTC TTTCTA ATG A SEQ ID NO: 21 containing a Xba1 restriction site. Theamplified TEF2 fragment was digested with Sac1/Xba1 and ligated intoSac1/Xba1 digested plasmid pRS416 (Sikorski and Hieter, 1989) asdescribed previously (Mumberg et al, 1995) resulting in plasmidpRS416-TEF2.

Example 6 Construction of Fused Divergent Constitutive TEF and TDH3Promoter Fragment

A divergent fusion fragment (FIG. 1) between TEF2 promoter and TDH3promoter was constructed starting from PRS416-TEF and PRS416-TDH3.

The 600 base pair TDH3 fragment was reamplified from PRS416-TDH3 usingthe forward primer 5′ TTGCGTATTGGGCGCTCTTCC GAG CTC AGT TTA TCA TTA TCAATA CTC GC SEQ ID NO: 22 containing the underlined overhang for fusionPCR to TEF2 fragment and the reverse primer 5′ AT GGATCC TCT AGA ATC CGTCGA AAC TAA GTT CTG SEQ ID NO: 23 containing the underlined BamH1restriction site. This resulted in a fragment ready for fusion to thebelow TEF2 fragment.

The 400 base pair TEF2 fragment including a 277 base pair spacerupstream of the Sad restriction site was reamplified from PRS416-TEF2using the forward primer 5′ AT GAATTC TCT AGA AAA CTT AGA TTA GAT TGCTAT GCT TTC SEQ ID NO: 24 containing the underlined EcoR1 restrictionsite and the reverse primer 5′ TGA TAA TGA TAA ACT GAG CTC GGA AGA GCGCCC AAT ACG CAA AC SEQ ID NO: 25 containing the underlined overhang forfusion to the TDH3 fragment. This resulted in a 680 base pair fragmentready for fusion to the TDH3 fragment.

The 680 base pair TEF2 fragment and the 600 base pair TDH3 fragmentswere joined together (fused) using fusion PCR with the forward primer 5′AT GAATTC TCT AGA AAA CTT AGA TTA GAT TGC TAT GCT TTC SEQ ID NO 37 andthe reverse primer 5′ AT GGATCC TCT AGA ATC CGT CGA AAC TAA GTT CTG SEQID NO: 26, resulting in the divergent fragment <=TEF2/TDH3=> (SequenceID NO 7).

Example 7 Construction of a Yeast Vector for Constitutive Expression ofPAL2 and C4H:AR2 Fusion Gene

The vector pESC-URA-PAL2-C4H:AR2 with divergent galactose induciblepromoters GAL1/GAL10 was sequentially digested with NotI and BsiWI toremove the GAL1/GAL10 promoters.

The divergent constitutive <=TEF2/TDH3=> promoter fragment (Example 6)was re-amplified with forward primer 5-GC CGTACG TCT AGA AAA CTT AGA TTAGAT TGC TAT GCT TTC-3 SEQ ID NO: 27 and reverse primer 5-ATT GCGGCCGCTCT AGA ATC CGT CGA AAC TAA GTT CTG-3 SEQ ID NO: 28. The resulting PCRproduct was sequentially digested with NotI and BsiWI and ligated intothe above vector without the GAM/Gall° fragment. This resulted in avector pESC-URA-TEF-PAL2-TDH3-C4H:AR2 with replaced promoters, fromGAM/Gall° to TEF2/TDH3 Sequence ID NO 8).

Example 8 Construction of a Yeast Vector for Constitutive Expression ofa TAL Gene

The vector pESC-URA-TAL with divergent galactose inducible promotersGAL1/GAL10 is sequentially digested with EcoRI and BamHI to remove theGAL1/GAL10 promoters.

The divergent constitutive <=TEF2/TDH3=> promoter fragment (Example 6)was sequentially digested with EcoR1 and BamH1 and ligated into theabove BamHI/EcoRI linearized pESC-URA-TAL vector without the GAL1/GAL10fragment. This resulted in a vector pesc-URA-TEF-TAL with replacedpromoters, from GAL1/Ga110 to TEF2/TDH3.

Example 9 Construction of a Yeast Vector for Constitutive ExpressionInduced of 4CL and VST1

The vector pESC-HIS-4CL-VST1 with divergent galactose induciblepromoters GAL1/GAL10 was sequentially digested with EcoR1 and BamH1 toremove the GAL1/GAL10 promoters.

The divergent constitutive <=TEF2/TDH3=> promoter fragment (Example 6)was sequentially digested with EcoR1 and BamH1 and ligated into theabove linearized vector without the GAL1/GAL10 fragment. This resultedin a vector pesc-HIS-TEF2-4CL-TDH3-VST1 with replaced promoters, fromGAL1/Gal10 to TEF2/TDH3 SEQ ID NO 9).

Example 10 Generation of a Strain with Constitutive Expression of thePhenylpropancid Pathway from Phenylalanine to Resveratrol in the YeastS. Cerevisiae

The transformation of the yeast cell was conducted in accordance withmethods known in the art, for instance by using lithium acetatetransformation method (Gietz and Schiestl, 1991). S. cerevisiae strainFS01528 (CEN.PK MATa ura3 His3) was co-transformed withpESC-URA-TEF-PAL2-TDH3-C4H:AR2 (example 7) andpesc-HIS-TEF2-4CL-TDH3-VST1 (example 9), and the transformed strain wasnamed FS09215. Transformants were selected on medium lacking uracil andhistidine and streak purified on the same medium.

Example 11 Generation of a Strain with Constitutive Expression of thePhenylpropancid Pathway from Tyrosine to Resveratrol in the Yeast S.Cerevisiae

The transformation of the yeast cell is conducted in accordance withmethods known in the art, for instance by using lithium acetatetransformation method (Gietz and Schiestl, 1991). S. cerevisiae strainFS01528 (CEN.PK MATa ura3 His3) is co-transformed with pESC-URA-TEF-TAL(example 8) and pesc-HIS-TEF2-4CL-TDH3-VST1 (example 9), to obtainstrain TEF2-TAL-TEF2-4CL-TDH3-VST1. Transformants are selected on mediumlacking uracil and histidine and streak purified on the same medium.

Example 12 HPLC Determination of Stilbenoids, Phenylpropanoids andEthanol

For quantitative analysis of cinnamic acid, trans-resveratrol andtrans-pinosylvin, samples were subjected to separation byhigh-performance liquid chromatography (HPLC) Agilent Series 1100 system(Hewlett Packard) prior to uv-diode-array detection at 2=306 nm. APhenomenex (Torrance, Calif., USA) Luna 2.5 micrometer C18 (100×2.00 mm)column was used at 60° C. As mobile phase a non linear S-shaped gradientof acetonitrile and milliQ water (both containing 50 ppm trifluoroaceticacid) was used at a flow of 0.8 ml/min. The S-shaped gradient profilewas from 10% to 100% acetonitrile in 5 minutes. The elution time wasapproximately 3.0 minutes for trans-resveratrol and 4.4 minutestrans-pinosylvin. Pure pinosylvin standard (>95% pure) was purchasedfrom ArboNova (Turku, Finland) and pure trans-resveratrol standard waspurchased from Sigma.

The grape-must was analysed for the content of ethanol by HPLC using anAminex HPX-87H ion-exclusion column (Bio-Rad, Hercules, Calif.) at 60°C., with 5 mM H2SO4 as a mobile phase at a flow rate of 0.6 ml/min. Theethanol was detected by a refractometer (Shodex RI-71).

Example 13 Generation of Biomass

Yeast strains FS01201 (CEN.PK 113-7D wild type non modified controlstrain) was kept on YPD agar plates with 20 g/l glucose. FS09215(genetically modified resveratrol producer from example 10) was kept onSC-HIS-URA agar plates with 20 g/l glucose.

The two yeast strains were grown in 10-16 500 ml shake flasks with 200ml DELFT medium (Verduyn et al, 1992) containing 45 g/l glucose, 30 g/lammonium sulphate, 14 g/l KH₂PO₄, and 1.5 g/l MgSO₄ for 4 days at 30° C.and 150 rpm. A paste of wet weight cells was collected (harvested) bycentrifugation at 3000 g for 5 minutes in 50 ml Sartorious tubes anddiscarding the supernatant after each round. After repetitive rounds ofcentrifugation 26 g wet weight was collected of strain FS01201 and 24 gwet weight of FS09215.

Example 14 Production of Red Wine from Grapes Using a S. CerevisiaeStrain with Constitutive Expression of the Phenylpropanoid Pathway fromPhenylalanine to Resveratrol

Commercially available seedless “Crimson” grapes (product of Brazil),were used to produce two red wine's: a control wine produced by usingstrain FS01201 and a stilbenoid-enriched wine by using strain FS09215 asdescribed in example 10. Said strain harbours a phenylpropanoid pathwaycomprising an oxygen-dependent cinnamate-4-hydroxylase (C4H) thatconverts cinnamic-acid in coumaric-acid. Hence, under theoxygen-deprived conditions that typically prevail in the anaerobicfermentation-process used for wine-making, said strain can only produceresveratrol if endogenous coumaric acid is present in the grape-pulp andgrape-must. With a lack of sufficient oxygen, however, said strain hasthe potential of producing pinosylvin, that is derived from cinnamicacid.

Preparation of Starter Culture

A starter culture of strain FS01201 and FS09215 was prepared by crushing2 kilo's of grapes and filtering the resulting grape pulp with aloose-woven cotton cloth (cheesecloth) and subsequently collecting thegrape juice in a sterile open plastic bucket. An aliquot of 500 ml ofgrape juice was enriched with 40 grams of glucose and divided into twoaliquots of 250 ml that were transferred to two sterile 500ml-shakeflasks. The shakeflasks were inoculated with approximately 2grams wet-weight of cells from either FS01201 or FS09215 as preparedaccording to example 13, and incubated for approximately 24 hours atroom temperature. Activity of yeast was indicated by formation of CO₂resulting in a foam-layer.

Primary Pulp Fermentation

A pulp of grapes was prepared by disrupting 16 kilo's of grapes with asemi-professional kitchen blender. The resulting grape-pulp was enrichedwith approximately 2 coffee-spoons of “yeast nutrient” (stimulates yeastgrowth) and 3 coffee-spoons of “pecto-enzymes” (breaks down pectin inthe grape skin). Both the pecto-enzymes and yeast nutrient were part ofa commercially available wine-making kit. Said enriched grape-pulp wasdivided into two equal aliquots, approximately 7 to 8 litre each, andtransferred into two 10 litre plastic round sterilized containers thatwere open at the top. A pulp-fermentation was initiated by adding thetotal amount of 250 ml of starter culture to the grape pulp, and mixingit well together with a large spoon; one bucket was inoculated withF501201, and the other with FS09215.

The progression of the pulp-fermentation was visually monitored on adaily basis, and the appearance of a foam-layer on the top, caused byCO₂ formation, indicated that the fermentation was successfully ongoing.The CO₂ formation caused the grape-pulp to float on top of thegrape-juice, and therefore the pulp was mixed with a large spoon on adaily basis as well. Formation of foam ceased after 9 days, and thegrape-pulp “fluidized”, which indicated that almost al grape-sugars wereconsumed and the pulp-fermentation was near its end. The grape pulp,therefore, was separated from the liquid fraction (containing the juiceand the yeast, i.e. the grape-must) by using a cheesecloth. For eachstrain approximately 5.5 to 6 litre of grape-must was collected. Thefermented juice was analyzed for the content of alcohol and stilbenoids.The FS01201- and FS09215 grape-must contained 84.01 g/l (i.e. 10.6 vol%) of ethanol and 79.65 g/l (i.e. 10.1 vol %) respectively. Thedevelopment of ethanol formation during the pulp fermentation is listedin table 2 below.

TABLE 2 Ethanol formation in primary pulp fermentation FS01201 FS09215g/l vol % g/l vol % Day 6 69.87 8.86 68.72 8.71 Day 7 83.69 10.61 81.810.37 Day 8 81.44 10.32 81.68 10.35 Day 9 84.01 10.65 79.65 10.10

Furthermore, no stilbenoids were found in either grape-must, however,low levels of cinnamic acid (1.05 mg/l) were determined in the FS09215grape-must. For the FS01201 and FS09215 pulp-fermentation, a total of1757 grams and 1780 grams wet-weight of grape-pulp was collectedrespectively. The grape pulp and approximately 1.5 litre of theremaining grape-must were stored at 4° C.

Secondary Grape-Must Fermentation

The fermentation was now continued with the grape-must. In order toenhance the alcohol percentage to a level that is usually found incommercial red wines (12- to 15 vol %), an aliquot of 3.5 litre ofgrape-must of either pulp fermentation was enriched with approximately340 g of commercially available sugar (“Dansukker”, dissolved and heatedin approximately 300 ml of water). The addition of the extra sugarcaused a dilution of the grape-must, resulting in a slight reduction ofethanol titers. Said enriched grape-must was transferred to a 5 litreglass-bottle that was stoppered with a “water-lock” to enable release ofCO2, but at the same time preventing contamination. After 4 days theethanol concentration rose to 99.46 g/l (i.e. 12.6 vol %) and 102.78 g/l(i.e. 13.0 volt). To enhance the ethanol concentration even further, asecond addition was made: 150 grams of sugar (“Dansukker”) was dissolvedinto 1400 ml of the previously stored non-enriched grape-must, which wasthen subsequently added to the fermentation glass-bottle. The totalvolume of the grape-must was now approximately 5 litres, and with thatalmost completely filled the glass bottle leaving no room for air in thetop of the bottle. The addition of the extra sugar caused a dilution ofthe grape-must, resulting in a slight reduction of ethanol titers. Aftera further 8 days, foam formation (i.e. CO₂ formation) ceased completely,indicating that the fermentation was finished and that all sugars weredepleted. The final ethanol concentration was 116.06 g/l (i.e. 14.7 vol%) and 109.64 g/l (i.e. 13.9 vol %), as listed in table 3.

TABLE 3 Ethanol-, phenylpropanoid- and stilbenoid formation in secondarygrape-must fermentation FS01201 FS09215 ethanol (g/l) ethanol (vol %)ethanol (g/l) ethanol (vol %) cinnamic acid (mg/l) pinosylvin (mg/l) Day10 82.39 10.44 85.21 10.80 1.19 0.62 Day 11 91.52 11.60 91.72 11.62 1.510.62 Day 12 99.73 12.64 98.2 12.45 not analyzed not analyzed Day 1399.46 12.61 102.78 13.03 not analyzed not analyzed Day 14 97.58 12.3792.04 11.67 1.36 0.57 Day 15 98.21 12.45 96.06 12.17 not analyzed notanalyzed Day 16 108.07 13.70 100.86 12.78 1.64 0.57 Day 17 109.45 13.87104.22 13.21 1.72 0.60 Day 18 120.48 15.27 111.6 14.14 1.63 0.60 Day 20121.79 15.44 111.84 14.17 1.76 0.57 Day 21 116.06 14.71 109.64 13.901.90 0.60

The reference strain FS01201 did neither producephenylpropanoid-intermediates nor stilbenoids. The chromatograms of thegrape-must of FS09215 contained a peak with a similar retention time aspinosylvin; indeed said peak with retention time of 4.4 minutes,displayed a UV-spectrum that was identical to pinosylvin. Similarly, thepresence of cinnamic acid was confirmed as well. Quantification of thepeaks indicated that the grape-must of strain FS09215 contained cinnamicacid and pinosylvin in final concentrations of 1.90 mg/l and 0.60 mg/lrespectively (Table 3). Neither resveratrol nor coumaric acid could bedetected, indicating that the activity of C4H was hampered by theanaerobic conditions. Furthermore, the lack of resveratrol and coumaricacid suggested that no endogenous coumaric acid was present in thegrape-must of the “Crimson” grapes used for this experiment.

In both fermentations, sediment settled on the bottom, which likelycomposed of small-size particle grape-residues and yeast cells. Saidsediment was isolated from the grape-must by siphoning, andapproximately 300 ml could be collected for either fermentation. Thesediments were stored at 4° C. until further analysis on stilbenoidcontent.

Analysis of Waste-Stream for the Presence of Stilbenoids

Approximately 125 grams of the grape pulp that was generated in theprimary pulp fermentation was extracted over night with 30 ml of ethylacetate (divided in three 50 ml Sartorious tubes) using a rotary unit atambient temperature (24° C.). The extraction tubes were covered withaluminium foil to avoid any light induced degradation of stilbenoids.The following day the extraction mixture was centrifuged at 3800×g for10 minutes, and the upper, yellow/greenish coloured, ethyl acetate wascollected and pooled into one tube. From the initial 30 ml ethylacetate, approximately 28 ml extract was collected for the FS1201grape-pulp suspension, and 30 ml of ethyl acetate for the FS09215grape-pulp suspension. Said ethyl acetate fractions were reduced involume by evaporation for 2 hours, using a freeze dryer, until a dryresidue was obtained.

The dry, dark coloured, residue was dissolved in 500 microlitre 50%ethanol which resulted in a solution that contained non-dissolved darkprecipitates. The solution was whirly-mixed and centrifuged at 13000×gfor 5 minutes and the supernatant was diluted 5-fold with 50% ethanol.Said procedure resulted in a clear yellowish solution that could be usedfor HPLC analysis.

The grape-pulp of the control strain FS01201 did neither containstilbenoids nor cinnamic- or coumaric acid (FIG. 6).

The chromatogram of the grape-pulp of FS09215 contained a peak with asimilar retention time as pinosylvin; indeed said peak with retentiontime of 4.4 minutes, displayed a UV-spectrum that was identical topinosylvin (FIGS. 6 and 7). Similarly, the presence of cinnamic acid wasconfirmed as well. Quantification of the peaks indicated that thegrape-pulp contained cinnamic acid and pinosylvin in concentrations of1.96 mg/kg pulp and 1.94 mg/kg pulp respectively. The total amount ofgrape-pulp recovered was 1780 grams, and can be considered as a primarywaste-stream generated with the production of 5 litre red wine.Therefore, the production of 5 litre of red wine led to a grape-pulpwaste-stream containing in total 1.780*1.96=3.49 mg cinnamic acid and1.780*1.94=3.45 mg pinosylvin. Hence, for 1 litre of red wine produced,3.49/5=0.70 mg of cinnamic acid- and 3.45/5=0.69 mg pinosylvin could berecovered from the pulp waste-stream.

Approximately 20 ml of sediment that was generated in the secondarygrape-must fermentation was extracted over-night with 10 ml of ethylacetate in a 50 ml Sartorious tube, using a rotary unit at ambienttemperature (24° C.). The extraction tubes were covered with aluminiumfoil to avoid any light induced degradation of stilbenoids. Thefollowing day the extraction mixture was centrifuged at 3800×g for 10minutes, and the upper, yellowish/greenish coloured, ethyl acetate wascollected and pooled into one tube. From the initial 10 ml of ethylacetate approximately 8 ml extract was recovered for both the FS1201-and FS9215 sediment suspension. Said ethyl acetate fractions werereduced in volume by evaporation for 2 hours using a freeze dryer untila dry residue was obtained. The dry, dark coloured, residue wasdissolved in 500 microlitre 50% ethanol which resulted in a solutionthat contained non-dissolved dark precipitates. The solution waswhirly-mixed and centrifuged at 13000×g for 5 minutes and thesupernatant was diluted 5-fold with 50% ethanol. Said procedure resultedin a clear yellowish solution that could be used for HPLC analysis.

The sediment of the control strain FS01201 did not contain stilbenoidsnor cinnamic- nor coumaric acid (FIG. 8).

The chromatogram of the sediment of FS09215 contained a peak with asimilar retention time as pinosylvin; indeed said peak with retentiontime of 4.4 minutes, displayed a UV-spectrum that was identical topinosylvin (FIGS. 8 and 7). Similarly, the presence of cinnamic acid wasconfirmed as well.

Quantification of the peaks indicated that the sediment containedcinnamic acid and pinosylvin in concentrations of 7.28 mg/l sediment and2.60 mg/L sediment respectively. The total amount of sediment recoveredwas approximately 300 ml, and can be considered as secondarywaste-stream generated with the production of 5 litre red wine.Therefore, the production of 5 litre of red wine led to a sedimentwaste-stream containing in total 0.3*7.28=2.18 mg cinnamic acid and0.3*2.60=0.78 mg pinosylvin. Hence, for 1 litre of red wine produced,2.18/5=0.44 mg of cinnamic acid- and 0.78/5=0.16 mg of pinosylvin couldbe recovered from the sediment waste stream.

Hence, for the production of 1 litre of red wine a total waste-streamwas produced from which a total amount of 0.70+0.44=1.14 mg cinnamicacid, and 0.69+0.16=0.85 mg pinosylvin could be recovered.

Example 15 Production of Red Wine from Grapes Using a S. CerevisiaeStrain with Constitutive Expression of the Phenylpropanoid Pathway fromTyrosine to Resveratrol

Commercially available seedless “Crimson” grapes (product of Brazil),are used to produce two red wine's: a control wine produced by usingstrain FS01201 and a stilbenoid enriched wine by using strainFS-TEF2-TAL-TEF2-4CL-TDH3-VST1 described in example 11. Said strainharbours a phenylpropanoid pathway comprising enzymes that do not useoxygen as substrate.

Hence, said strain can produce resveratrol under the oxygen-deprivedconditions that typically prevail in the anaerobic fermentation-processused for wine-making.

Preparation of Starter Culture

A starter culture of strain FS01201 and FS-TEF2-TAL-TEF2-4CL-TDH3-VST1is prepared by crushing 2 kilo's of grapes and filtering the resultinggrape pulp with a loose-woven cotton cloth (cheesecloth) andsubsequently collecting the grape juice in a sterile open plasticbucket. An aliquot of 500 ml of grape juice is enriched with 40 grams ofglucose and divided into two aliquots of 250 ml that are transferred totwo sterile 500 ml-shakeflasks. The shakeflasks are inoculated withapproximately 2 grams wet-weight of either FS01201 orFS-TEF2-TAL-TEF2-4CL-TDH3-VST1 as prepared according to example 1, andincubated for approximately 24 hours at room temperature. Activity ofyeast is indicated by formation of CO₂ resulting in a foam-layer.

Primary Pulp Fermentation

A pulp of grapes is prepared by disrupting 16 kilo's of grapes with asemi-professional kitchen blender. The resulting grape-pulp is enrichedwith approximately 2 coffee-spoons of “yeast nutrient” (stimulates yeastgrowth) and 3 coffee-spoons of “pecto-enzymes” (breaks down pectin inthe grape skin). Both the pecto-enzymes and yeast nutrient are part of awine-making kit that is commercially available. Said enriched grape-pulpis divided into two equal aliquots, approximately 7 to 8 litre each) andtransferred into a 10 litre plastic round sterilized container that isopen at the top. A pulp-fermentation is initiated by adding the totalamount of 250 ml of starter culture to the grape pulp, and mixing itwell together with a large spoon; one bucket is inoculated with F501201,and the other with FS-TEF2-TAL-TEF2-4CL-TDH3-VST1.

The progression of the pulp-fermentation is visually monitored on adaily basis, and the appearance of a foam-layer on the top, caused byCO₂ formation, indicates that the fermentation is successfully ongoing.The CO₂ formation causes the grape-pulp to float on top of thegrape-juice, and therefore the pulp is mixed with a large spoon on adaily basis as well. The pulp-fermentation is near its end when almostal grape-sugars are consumed, which is indicated by cessation offoam-formation and “fluidizing” of the grape-pulp. The grape pulp isthen separated from the liquid fraction (containing the juice and theyeast, i.e. the grape-must) by using a cheesecloth. For each strainapproximately 5.0 to 6 litres of grape-must is collected and analyzedfor the content of alcohol and stilbenoids. The grape pulp andapproximately 1.5 litres of the remaining non-enriched grape-must arestored at 4° C.

Secondary Grape—Must Fermentation

The fermentation is now continued with the grape-must. In order toenhance the alcohol percentage to a level that is usually found incommercial red wines (12- to 15 vol %), an aliquot of 3.5 litre ofgrape-must of either pulp fermentation is enriched with approximately340 g of commercially available sugar (“Dansukker”, dissolved and heatedin approximately 300 ml of water). The addition of the extra sugarcauses a dilution of the grape-must, resulting in a slight reduction ofethanol titers. The enriched grape-must is transferred to a 5 litreglass-bottle that is stoppered with a “water-lock” to enable release ofCO₂, but at the same time preventing contamination. When the ethanolconcentration reaches a level in between 12- to 13 vol %., a secondsugar-addition is made to enhance the ethanol concentration evenfurther: 150 grams of sugar (“Dansukker”) is dissolved into 1400 ml ofthe previously stored non-enriched grape-must, which is thensubsequently added to fermentation glass-bottle. The total volume of thefermentation broth is now approximately 5 litres, and with that almostcompletely fills the glass bottle leaving no room for air in the top ofthe bottle. The addition of the extra sugar causes a dilution of thegrape-must, resulting in a slight reduction of ethanol titers. Thefermentation is finished when all sugars are depleted, and is indicatedby a complete cessation of foam formation (i.e. CO₂ formation), and thefinal ethanol concentration is in between 14- to 15 vol %. For eachstrain the grape-must is analyzed for the content of alcohol andstilbenoids.

In both fermentations sediment settles on the bottom, which likelycomposes of small-sized particle grape-residues and yeast cells. Saidsediment is isolated from the grape-must by siphoning. The sediments arestored at 4° C. until further analysis on stilbenoid content.

Analysis of Waste-Stream for the Presence of Stilbenoids

Approximately 125 grams of the grape pulp that is generated in theprimary pulp fermentation is extracted over night with 30 ml of ethylacetate (divided in three 50 ml Sartorious tubes) using a rotary unit atambient temperature (24° C.). The extraction tubes are covered withaluminium foil to avoid any light induced degradation of stilbenoids.The following day the extraction mixture is centrifuged at 3800×g for 10minutes, and the upper, yellow/greenish coloured, ethyl acetate iscollected and pooled into one tube. Said ethyl acetate fractions arereduced in volume by evaporation for 2 hours, using a freeze dryer,until a dry residue is obtained. The dry, dark coloured, residue isdissolved in 500 microlitre 50% ethanol which results in a solution thatcontains non-dissolved dark precipitates. The solution is whirly-mixedand centrifuged at 13000×g for 5 minutes and the supernatant is diluted5-fold with 50% ethanol. Said procedure results in a clear yellowishsolution that can be used for HPLC analysis.

The grape-pulp can be considered as a primary waste-stream generatedwith the production of 5 litre wine, and the resveratrol that can berecovered from the pulp can be expressed in terms of production of 1litre red-wine.

Approximately 20 ml of sediment that is generated in the secondarygrape-must fermentation is extracted over night with 10 ml of ethylacetate in a 50 ml Sartorious tube, using a rotary unit at ambienttemperature (24° C.). The extraction tubes are covered with aluminiumfoil to avoid any light induced degradation of stilbenoids. Thefollowing day the extraction mixture is centrifuged at 3800×g for 10minutes, and the upper, yellowish/greenish coloured, ethyl acetate iscollected and pooled into one tube. Said ethyl acetate fractions arereduced in volume by evaporation for 2 hours, using a freeze-dryer,until a dry residue is obtained.

The dry, dark coloured, residue is dissolved in 500 microlitre 50%ethanol which results in a solution that contains non-dissolved darkprecipitates. The solution is whirly-mixed and centrifuged at 13000×gfor 5 minutes and the supernatant is diluted 5-fold with 50% ethanol.Said procedure results in a clear yellowish solution that can be usedfor HPLC analysis.

The sediment can be considered as a secondary waste-stream generatedwith the production of 5 litre wine, and resveratrol that can berecovered from the sediment can be expressed in terms of production of 1litre red-wine.

Hence, for the production of 1 litre of red wine the total amount ofresveratrol that can be recovered from the waste-stream can be found bysummation of the amount of the resveratrol present in both the primary-and secondary waste-stream.

Example 16 Isolation of Genes Encoding SAM8, 4CL2 and VST1

The codon optimized SAM8 gene encoding Saccharotrix espaniensis Tyrosineammonia lyase (Berner et al, 2006) (SEQ ID NO 38) for expression in S.cerevisiae was synthesized by GenScript Corporation (Piscataway, N.J.).The synthetic Sam8 gene was delivered inserted in E. coli pUC57 vectorflanked by EcoRI and SpeI restriction sites. The synthetic gene waspurified from the pUC57 vector by EcoRI/SpeI restriction and purifiedfrom agarose gel using the QiaQuick Gel Extraction Kit (Qiagen).

4-coumarate:coenzymeA ligase (4CL2) (Hamberger and Hahlbrock 2004;Ehlting et al., 1999) (SEQ ID NO 39) was isolated via PCR from A.thaliana cDNA (BioCat, Heidelberg, Germany) using the primers, Forward5′GCGAATTCTTATGACGACACAAGATGTGATAGTCAATGAT SEQ ID NO 40 with theunderlined restriction sequence for ECOR1, and Reverse5′GCACTAGTATCCTAGTTCATTAATCCATTTGCTAGTCTTGC SEQ ID NO 41 with theunderlined restriction site for SpeI.

The codon optimized VST1 gene encoding Vitis vinifera (grapevine)resveratrol synthase (Hain et al., 1993) (SEQ ID NO 42) for expressionin S. cerevisiae was synthesized by GenScript Corporation (Piscataway,N.J.). The synthetic VST1 gene was delivered inserted in E. coli pUC57vector flanked by BamH1 and Xho1 restriction sites. The synthetic genewas purified from the pUC57 vector by BamH1/Xho1 restriction andpurified from agarose gel using the QiaQuick Gel Extraction Kit(Qiagen).

Example 17 Construction of a Yeast Vector for Galactose InducedExpression of SAM8

The EcoRI/SpeI digested SAM8 product, isolated as described in example16, was ligated into EcoRI/SpeI digested pESC-URA vector (Stratagene),resulting in vector pESC-URA-SAM8. Two different clones of pESC-URA-SAM8were sequenced to verify the sequence of the cloned gene.

Example 18 Construction of a Yeast Vector for Galactose InducedExpression of 4CL2 and VST1

The gene encoding 4CL2 was isolated using the primers as described inexample 16. The amplified 4CL2 PCR-product was digested with EcoR1/Spe1and ligated into EcoR1/Spe1 digested pESC-HIS vector (Stratagene),resulting in vector pESC-HIS-4CL2. Two different clones of pESC-HIS-4CL2were sequenced to verify the sequence of the cloned gene.

The gene encoding VST1 was isolated as described in example 16. Theamplified synthetic VST1 gene was digested with BamH1/Xho1 and ligatedinto BamH1/Xho1 digested pESC-HIS-4CL2. The resulting plasmid,pESC-HIS-4CL2-VST1, contained the genes encoding 4CL2 and VST1 under thecontrol of the divergent galactose induced <=GAL1/GAL10=> promoters. Thesequence of the gene encoding VST1 was verified by sequencing of twodifferent clones of pESC-HIS-4CL2-VST1.

Example 19 Construction of Strong Constitutive Promoter Fragment TDH3

The 600 base pair TDH3 (GPD) promoter was amplified from S. cerevisiaegenomic DNA using the forward primer 5′GC GAGCTC AGT TTA TCA TTA TCA ATACTC GCC ATT TCA AAG SEQ ID NO 43 containing a Sad restriction site andthe reverse primer 5′-CG TCTAGA ATC CGT CGA AAC TAA GTT CTG GTG TTT TAAAAC TAA AA SEQ ID NO 44 containing a Xba1 restriction site. Theamplified TDH3 fragment was digested with Sac1/Xba1 and ligated intoSac1/Xba1 digested plasmid pRS416 (Sikorski and Hieter, 1989) asdescribed previously (Mumberg et al, 1995) resulting in plasmidpRS416-TDH3.

Example 20 Construction of Constitutive Strong Promoter Fragment TEF2

The 400 base pair TEF2 promoter was amplified from S. cerevisiae genomicDNA using the forward primer 5′-GC GAGCTC ATA GCT TCA AAA TGT TTC TACTCC TTT TTT ACT CTT SEQ ID NO 45 containing a Sac1 restriction site andthe reverse primer 5′-CG TCTAGA AAA CTT AGA TTA GAT TGC TAT GCT TTC TTTCTA ATG A SEQ ID NO 46 containing a Xba1 restriction site. The amplifiedTEF2 fragment was digested with Sac1/Xba1 and ligated into Sac1/Xba1digested plasmid pRS416 (Sikorski and Hieter, 1989) as describedpreviously (Mumberg et al, 1995) resulting in plasmid pRS416-TEF2.

Example 21 Construction of Fused Divergent Constitutive TEF and TDH3Promoter Fragment

A divergent fusion fragment between TEF2 promoter and TDH3 promoter wasconstructed starting from PRS416-TEF and PRS416-TDH3.

The 600 base pair TDH3 fragment was reamplified from PRS416-TDH3 usingthe forward primer 5′ TTGCGTATTGGGCGCTCTTCC GAG CTC AGT TTA TCA TTA TCAATA CTC GC SEQ ID NO 47 containing the underlined overhang for fusionPCR to TEF2 fragment and the reverse primer 5′ AT GGATCC TCT AGA ATC CGTCGA AAC TAA GTT CTG SEQ ID NO 48 containing the underlined BamH1restriction site. This resulted in a fragment ready for fusion to thebelow TEF2 fragment.

The 400 base pair TEF2 fragment including a 277 base pair spacerupstream of the Sad restriction site was reamplified from PRS416-TEF2using the forward primer 5′ AT GAATTC TCT AGA AAA CTT AGA TTA GAT TGCTAT GCT TTC SEQ ID NO 49 containing the underlined EcoR1 restrictionsite and the reverse primer 5′ TGA TAA TGA TAA ACT GAG CTC GGA AGA GCGCCC AAT ACG CAA AC SEQ ID NO 50 containing the underlined overhang forfusion to the TDH3 fragment. This resulted in a 680 base pair fragmentready for fusion to the TDH3 fragment.

The 680 base pair TEF2 fragment and the 600 base pair TDH3 fragmentswere joined together (fused) using fusion PCR with the forward primer 5′AT GAATTC TCT AGA AAA CTT AGA TTA GAT TGC TAT GCT TTC SEQ ID NO 51 andthe reverse primer 5′ AT GGATCC TCT AGA ATC CGT CGA AAC TAA GTT CTG SEQID NO 52, resulting in the divergent fragment <=TEF2/TDH3=> (SEQ ID NO53).

Example 22 Construction of a Yeast Vector for Constitutive Expression ofSam8

The vector pESC-URA-Sam8 with divergent galactose inducible promotersGAL1/GAL10 was sequentially digested with EcoRI and BamHI to remove theGAL1/GAL10 promoters.

The divergent constitutive <=TEF2/TDH3=> promoter fragment (Example 21)was re-amplified with forward primer 5′ AT GGATCC TCT AGA AAA CTT AGATTA GAT TGC TAT GCT TTC SEQ ID NO 54 and reverse primer 5′AT GAATTCTCTAGA ATC CGT CGAAACTAAGTTCTGG SEQ ID NO 55.

The resulting PCR product was sequentially digested with ECORI and BamH1and ligated into the above ECORI/BamHI digested vector (pESC-URA-Sam8)without the GAL1/Gal10 fragment. This resulted in a vectorpESC-URA-TDH3-Sam8 with replaced promoters, from GAL1/Gal10 to TEF2/TDH3(SEQ ID NO 56).

Example 23

Construction of a yeast vector for constitutive expression induced of4CL2 and VST1

The vector pESC-HIS-4CL2-VST1 with divergent galactose induciblepromoters GAL1/GAL10 was sequentially digested with EcoR1 and BamH1 toremove the GAL1/GAL10 promoters.

The divergent constitutive <=TEF2/TDH3=> promoter fragment (Example 21)was sequentially digested with EcoR1 and BamH1 and ligated into theabove linearized vector without the GAL1/GAL10 fragment. This resultedin a vector pesc-HIS-TEF2-4CL2-TDH3-VST1 with replaced promoters, fromGAL1/Gal10 to TEF2/TDH3 (SEQ ID NO 57).

Example 24 Generation of Strain with Constitutive Expression of thePathway to Resveratrol in the Yeast S. Cerevisiae

The transformation of the yeast cell was conducted in accordance withmethods known in the art, for instance by using lithium acetatetransformation method (Gietz and Schiestl, 1991). S. cerevisiae strainFS01528 (CEN.PK MATa ura3 His3) was co-transformed withpESC-URA-TDH3-SaM8 (example 22) and pesc-HIS-TEF2-4CL2-TDH3-VST1(example 23), and the transformed strain was named FS-SAM8-4CL2-VST1.Transformants were selected on medium lacking uracil and histidine andstreak purified on the same medium.

Example 25 Generation of Biomass

Batch fermentation of FS-SAM8-4CL2-VST1 were carried out in order togenerate wet biomass for inoculation of the wine fermentation. Thefermentation was carried out in a Sartorius Biostat B plus fermentorunder aerobic conditions. The working volume was 1 L, agitation was 1000rpm, and air flow was set to 1.5 vvm. Temperature and pH were 30° C. and5.5, respectively. The fermentation was inoculated to an initial OD of0.0005. The composition is described in the following:

Media:

Compound Concentration [g/L] Glucose 165 Urea 22.72 KH2PO4 30 MgSO4 5Vitamin solution 10 mL* Trace metal solution 10 mL* Antifoam 100 μLVitamin solution

Concentration [g/L] Biotin 0.05 Calcium panthotenate 1 Nicotinic acid 1Myo-inositol 25 Thiamine HCl 1 Pyridoxal HCl 1 Para-aminobenzoic acid0.2Trace metal solution

Concentration [g/L] EDTA 15 ZnSO4 × 7H2O 4.5 MnCl2 × 2H2O 1 CoCl2 × 6H2O0.3 CuSO4 × 5H2O 0.3 Na2MoO4 × 2H2O 0.4 CaCl2 × 2H2O 4.5 FeSO4 × 7H2O 3H3BO3 1 KI 0.1

At the end of the fermentation, when all glucose was depleted, thebiomass was harvested into Falcon tubes by centrifugation. Four 50 mLsterile Falcon tubes were used and the cells were harvested using 5consecutive centrifuge runs at 4° C. with 4000 rpm for five minutes. Thecentrifuge used was a Satorius Sigma 3-16K including a swing out rotorwith for buckets. The supernatant was each discarded after each run. Thefour tubes contained approximately 70 g of wet biomass, which was usedas inoculum for the wine fermentation.

Example 26 Resveratrol Production by Fermenting Wine

Objective: To evaluate the relative efficacy of producing resveratrol bywine fermentation using wine juice concentrate as plant materialcompared to commercially available wine yeast,

Two identical wine making kits procured from Winexpert Incorporated ofCanada were used as the basis of making a comparison between thecommercially available yeast Saccharomyces bayanus (Lalvin EC1118), thecontrol strain, for wine making versus FS-SAM8-4CL2-VST1. The kit usedwas the “Selection Original—Barolo Style.” This style utilizes theNebbiolo red grape as the basis of the grape juice concentrate. Thisconcentrate is preserved with suphur dioxide, citric acid, malic acid,tartaric acid, and diammonium phosphate. Also supplied in the kit aresingle packages of a premeasured amount of bentonite for use as aclarifying agent, potassium metabisuphite as a stabilizer, and oak chipsused for flavoring. The package of oak chips was not used in thisexperiment for either fermentation. Kit designated Lot 07318080147 wasused for fermentation of Saccharomyces bayanus (Lalvin EC1118) and Lot07318080172 was for fermentation of FS-SAM8-4CL2-VST1.

Each of two kits was treated similarly except for the inoculum. For thewine fermentation using the control strain five grams of yeast (LalvinEC1118 of Lallemand Inc.) were used, whereas for FS-SAM8-4CL2-VST1 18 gof wet biomass, which was approximately 5 g/L dry weight, was used asinoculum.

Before inoculation, all equipment and containers being used were firstsanitized with a solution of metabisulfite (˜50 grams in 4 liters).Spring water (“Deerpark”) was used for all solutions. Two liters of warmwater (˜300° C.) were added to a clean 30 liter plastic carboy containerand stirred vigorously while slowly sprinkling in the contents of thebentonite package until fully wetted and dispersed; for approximatelyone minute. The grape juice concentrate (15 L) were filled into thecontainers. The package of grape juice concentrate was washed with 4liters, which was afterwards added to container. The final containervolume was adjusted to 23 liters with cool. The oak chip packageintended to be used as a flavoring enhancer was not used in either thecontrol or treatment groups. With the juice solution at about roomtemperature (˜20.0° C.), the package of Lalvin yeast was sprinkled ontop of the control juice solution and FS-SAM8-4CL2-VST1 was poured intosolution. Each container (primary fermentor) was covered with anair-lock. The wine was fermented for 50 days. Samples of wine and mashwere taken at the end of the fermentation and placed into a sealedplastic cup and stored frozen until analyses.

Example 27 Extraction of Resveratrol from Wine-Mash

Two duplicate samples of the mash from the control- and treatment groupswere evaluated for their content of stilbenoids, hereafter referred toas control A, control B, treatment A and treatment B. Aliquots of 50 mlof either samples, containing a mixture of mash and wine, werecentrifuged for 10 minutes at 3500XG at 10° C., after which thesupernatant was discarded. Hereafter, the wet weight content of mash was12.57 g for control A, 14.03 g for control B, 11.63 g for treatment Aand 12.52 g for treatment B. Next, 10 ml of 99% ethyl acetate was addedand whirly-mixed at room temperature on an automated whirly mixer for 2hours at 2500 rpm. Then samples were centrifuged for 10 minutes at3500×g at 10° C., and the upper, yellow/reddish coloured, ethyl acetatewas collected reduced in volume by evaporation in a freeze-dryer. Afterapproximately 2 hours a dry reddish residue was obtained, which wascarefully resuspended in 120 μl 20% ethanol and resulted in a solutionthat contained non-dissolved dark precipitates. The solution was,therefore, whirly-mixed and centrifuged at 13000×g for 5 minutes. Thesupernatant now consisted of a clear reddish 20%-ethanol solution, whichwas diluted 100-fold further in two steps of 10-fold; the first dilutionstep was rendered in 20%-ethanol whereas Millipore water was used forthe subsequent second 10-fold dilution step. Samples were then ready tobe analyzed by HPLC.

Example 28 HPLC Determination of Stilbenoids and Phenylpropanoids

For quantitative analysis of coumaric acid, cinnamic acid,trans-resveratrol and trans-pinosylvin, samples were subjected toseparation by high-performance liquid chromatography (HPLC), using aHPLC-system from Dionex, prior to UV-diode-array detection at 1=306 nm.A Phenomenex (Torrance, Calif., USA) Gemini C6-Phenyl, 3 micron(100×3.00 mm) column was used at 35° C. The method consisted of a lineargradient of methanol and millipore water (both containing 50 ppmtrifluoroacetic acid), at a flow rate of 0.5 ml/min. The gradientprofile was linear from 20% methanol to 100% methanol over 20 min. Theelution times were 7.5 min. for coumaric acid, 10.1 min. fortrans-resveratrol, 11.8 min. for cinnamic acid and 14.0 min forpinosylvin.

Example 29 Concentration of Resveratrol in the Mash

The chromatograms of both the control group and the treatment groupcontained all a peak with a similar retention time as resveratrol (9.9minutes) and with an UV spectrum that resembled the UV spectrum ofresveratrol. Quantification of the peak indicated that the resveratrolcontent in the mash was 0.33 mg/kg for control A, 0.48 mg/kg for controlB, giving an average of 0.41 mg/kg for the control group. The mash oftreatment A contained 0.80 mg/kg and treatment B contained 0.73 mg/kg,giving an average resveratrol content of 0.77 mg/kg for the treatmentgroup. Hence, on average, the mash of the treatment group contained 89%more resveratrol than the mash of the control group.

It can, therefore, be concluded that the use of resveratrol-producingyeast in a wine fermentation process has led to a substantial enrichmentof the resveratrol in the mash.

In this specification, unless expressly otherwise indicated, the word‘or’ is used in the sense of an operator that returns a true value wheneither or both of the stated conditions is met, as opposed to theoperator ‘exclusive or’ which requires that only one of the conditionsis met. The word ‘comprising’ is used in the sense of ‘including’ ratherthan in to mean ‘consisting of’. All prior teachings acknowledged aboveare hereby incorporated by reference. No acknowledgement of any priorpublished document herein should be taken to be an admission orrepresentation that the teaching thereof was common general knowledge inAustralia or elsewhere at the date hereof.

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1. A method for the production of a mixture comprising stilbenoids,comprising extracting resveratrol or pinosylvin from a solids wastematerial separated from a fermentation of plant material conducted usinga genetically modified yeast strain having a metabolic pathway producingthe mixture comprising stilbenoids.
 2. The method of claim 1, furthercomprising the preliminary steps of conducting said fermentation ofplant material using the genetically modified yeast strain having ametabolic pathway producing the mixture comprising stilbenoids andseparating a solids waste material from said fermentation.
 3. The methodof claim 1, wherein the fermentation is a fermentation of fruit musttogether with or separated from pommace.
 4. The method of claim 1,wherein the fermentation is a fermentation of pommace separated fromfruit must.
 5. The method of claim 2, wherein the fruit is grape, appleor pear.
 6. The method of claim 1, wherein the fermentation is a beermaking fermentation.
 7. The method of claim 1, wherein the geneticallymodified yeast strain is genetically modified Saccharomyces yeast. 8.The method of claim 1, further comprising the step of recoveringresveratrol or pinosylvin.